Differential gene regulation in DAPT-treated Hydra reveals candidate direct Notch signalling targets

In Hydra, Notch inhibition causes defects in head patterning and prevents differentiation of proliferating nematocyte progenitor cells into mature nematocytes. To understand the molecular mechanisms by which the Notch pathway regulates these processes, we performed RNA-seq and identified genes that are differentially regulated in response to 48 h of treating the animals with the Notch inhibitor DAPT. To identify candidate direct regulators of Notch signalling, we profiled gene expression changes that occur during subsequent restoration of Notch activity and performed promoter analyses to identify RBPJ transcription factor-binding sites in the regulatory regions of Notch-responsive genes. Interrogating the available single-cell sequencing data set revealed the gene expression patterns of Notch-regulated Hydra genes. Through these analyses, a comprehensive picture of the molecular pathways regulated by Notch signalling in head patterning and in interstitial cell differentiation in Hydra emerged. As prime candidates for direct Notch target genes, in addition to Hydra (Hy)Hes, we suggest Sp5 and HyAlx. They rapidly recovered their expression levels after DAPT removal and possess Notch-responsive RBPJ transcription factorbinding sites in their regulatory regions Overall design: We aimed to identify the transcriptional target genes of Notch signalling by comparing DAPT-treated animals to control ones at three different time points. Six biological repeats were used.

在水螅（Hydra）中，抑制Notch信号通路（Notch pathway）会引发头部模式建成缺陷，并阻断增殖的刺细胞祖细胞向成熟刺细胞的分化过程。为解析Notch通路调控上述过程的分子机制，我们开展了RNA测序（RNA-seq）实验，鉴定出经Notch抑制剂DAPT处理动物48小时后差异表达的基因。为筛选Notch信号的直接调控候选靶点，我们检测了后续恢复Notch活性过程中的基因表达变化，并通过启动子分析，在Notch响应基因的调控区域中鉴定RBPJ转录因子（RBPJ）结合位点。通过分析现有单细胞测序（single-cell sequencing）数据集，我们明确了受Notch调控的水螅基因的表达模式。经上述系列分析，我们完整勾勒出Notch信号通路在水螅头部模式建成与间质细胞分化过程中所调控的分子通路全貌。除水螅HyHes外，我们提出Sp5与HyAlx可作为Notch直接靶基因的核心候选靶点：二者在撤除DAPT后可快速恢复其表达水平，且其调控区域中携带有具有Notch响应性的RBPJ转录因子结合位点。实验整体设计：本研究旨在通过在三个不同时间点对比DAPT处理组与对照组动物的转录组，鉴定Notch信号的转录靶基因，共设置6次生物学重复。